The mysteries of sperm function

Tenacious and
audacious in equal measure, Associate Professor Mark Baker is seeking to crack the genetic code
of male infertility.

Diagnosing male factor infertility – which accounts for half of all Assisted Reproduction Technologies (ART) procedures in Australia – is akin to solving a 5000 odd-piece jigsaw puzzle. Some pieces or answers, like reduced sperm
number and motility, are right in front of you; they're big and obvious, and usually the first to be picked up and put down by novice game players. They frame the puzzle and make it easier to work inward. Then there are some pieces, like genetic differences, that are much smaller in size or even hidden;
they're not your typical go-tos and they're not easy to place. But they are necessary to complete the picture.

Associate Professor Mark Baker, touted as a pioneer of sperm proteomics, is leading worldwide efforts to explore and understand the latter.

I study the structure and function of proteomes

"These are the entire sets of proteins that are expressed by genomes."

The professional researcher, who was awarded a National Health and Medical Research Council Career Development Fellowship in early 2012, uses mass spectrometry to illustrate the chemical composition of larger molecules and cells. Spanning the life sciences, medicine and engineering arenas, this work
is housed in a state-of-the-art laboratory at the University of Newcastle.

"The implications of our research are widespread," Mark affirms.

"About 1 in 15 men are infertile."

"Medicare-funded statistics point to an IVF success rate of just 22 per cent, which is equally concerning."

"It's important we develop more efficient and effective ways of finding the best sperm."

Divide and
conquer

Taking a "roundabout route" to get to this point, Mark began his research career in the cancer field. The energetic educator and innovator undertook a PhD at Melbourne's Monash University in 1999, principally using it to investigate the relationship between chemotherapy and cell death.

"We wanted to look at what the chemical treatment is doing to the body," he asserts.

"It's basically a poison."

Seeking to gain a more intricate understanding of the cell cycle during his three-year probe, Mark closely observed the mechanism by which microscopic units were dying during the chemotherapy process.

"Cells that are dividing are more susceptible to cell death than cells that aren't dividing," he explains.

"Cancers can be destroyed as the cells die, but that happens to be how people lose their hair and the lining of the stomach as well."

Back to basics

Mark relocated to Newcastle after his PhD was conferred in 2002, linking up with Laureate Professor John Aitkin and the University's influential Reproductive Science Group. Inspired by new technologies at the time, the creative collaborator looked to put an unusual twist on his PhD theme.

"Proteomics allowed us to compare the difference between normal biological cells and cancer cells," he states.

"We can do the same thing with male reproductive cells - compare the difference between those belonging to fertile men and those belonging to infertile men and find out what's missing."

"It's a fascinating question."

From 2002-2011, Mark also undertook some basic science research on the behaviours and movement patterns of sperm. This time exploring the differences between mature cells and immature cells, the expert sought to understand the "hows" and "whys" of their storage and travel.

"We found that a sperm is immotile in the testes and cannot recognise the egg until it reaches the bottom of the epididymis," he illuminates.

"We found that immature sperm undergo changes in their phosphorylation status too."

"Some of the motor proteins become 'oiled' and this enables the tail to become motile, which is necessary for fertilisation."

"Essentially, the whole head and surface of the sperm are remodelled from the early regions to the later regions."

Spot the
difference

Still endeavouring to define the differences between the sperm of fertile men and the sperm of infertile men, Mark is aiming to one day soon diagnose and prognose 80-90% of male infertility cases. Working on the proviso that "the more cases looked at, the more that is found," the ambitious academic
is in the process of analysing more than 100 samples locked away in his laboratory.

"At the moment we can probably detect 30%," he declares.

"Some of the guys are missing proteins in the head area, which means they're unable to recognise and bind with the egg."

"Others are missing a protein in the neck area, so the head and tail actually fall apart over a period of time."

"Proteins in the tail area are stopping the sperm from being normally motile as well."

Believing such patterns will "become repetitive" with enough data, Mark is hoping to have a diagnostic framework completed in 2-3 years' time.

"The hardest thing for us is obtaining donations," he acknowledges.

"At the moment, we've had to go to Brazil."

"We need interested, local men to come forward."

"Within a week we can do five samples and determine what's missing within them, so it doesn't take very long."

At the same time, Mark is collaborating with neurology expert Dr Neil Spratt on a clinical stroke research project. Again attempting to figure out the "whys" behind the "what is," the duo is seeking to compare the difference between normal cerebral spinal fluid and cerebral spinal fluid that belongs
to patients who've had a "minor stroke."

"In a period of 24 hours while they're recovering, the latter group will often have another, more major stroke," the reproductive scientist elucidates.

"It's killing a lot of people."

Already identifying a handful of molecules responsible for causing the second, lethal stroke, the pair is eager to move on to the prevention stage and develop a number of single-targeted inhibitors.

Changing tactics
for changing times

Keen to move past mere motility and morphology when it comes to analysing semen samples, Mark is pushing for big transformations in the way suspected infertilities are investigated in Australia. The University of Newcastle Research Fellow has been doing so since he made the switch to translational
research in 2012.

"Females have to undergo invasive laparoscopies and pelvic examinations," he reveals.

"We want the focus to go instantly to the male."

"It's so easy to get an ejaculate and bring it to the lab."

"Within an hour we can have a diagnosis as to whether the couple can conceive or not."

Kicking goals

Dr Mark Baker has picked up the pace of sperm cell research
by harnessing the power of proteomics.

There is a competitive side to Dr Mark Baker, who likes to keep fit and, at 37, still enjoys the challenge of matching strides with younger opponents on the soccer field. But the race Baker is most keen to win is the one to develop a male version of the contraceptive pill – and the proteomics
expert believes his research team at the University of Newcastle has a real chance of achieving that goal.

"We are the world leaders in sperm proteomics," points out Baker, who was the first researcher to create a complete inventory of the proteins in sperm and has established a state-of-the-art proteomics laboratory at the University. "The work we do is part of a worldwide effort but we are recognised
as pioneers and the most progressive group in our field."

Baker joined the University's influential Reproductive Science Group, led by Laureate Professor John Aitken, after being awarded a PhD from Monash University in 2002. Aitken, who specialises in sperm cell biology, recognised the potential in combining their areas of expertise towards the common goal
of solving the mysteries of male fertility.

Whilst Baker's ultimate goal is to develop a male contraceptive, his research has equal potential to assist men who are infertile, as it is necessary to understand the mechanisms of infertility in order to mimic the condition with a male contraceptive. In identifying and publishing the sperm proteome,
Baker advanced the number of known proteins from less than 40 to more than 1200. He and the research team have subsequently narrowed down that list of proteins to a handful that can potentially be targeted by fertility drugs.

"One of the important criteria is that the protein is found only in the testes, because if you are designing a drug that inhibits a protein you don't want it to affect other parts of the body," Baker explains. "So, we have identified a number of proteins that we know are both unique to the testes
and are vital for fertility. We are now moving into the next phase of the research, which is designing a drug to target them."

Baker's work has attracted the attention of Bayer (formerly Bayer Schering Pharma), one of the first companies to market the contraceptive pill back in the 1960s. He has also established collaborations with leading international research groups in the field.

"The implications of our research are widespread when you consider that about half of all pregnancies in Australia are unwanted or unplanned and that there are about 250 abortions for every 1000 live births," Baker asserts. "There are also thousands of women who suffer side effects from contraception
and there is an inextricable link between poverty and high birth rates in developing counties. As you can imagine, there is a real global imperative to improve options by developing a male contraceptive. At the same time, up to 10 per cent of the male population is infertile, so we want to be able to
help them as well."

Baker has contributed his expertise in identifying the protein structure of cells to other interesting collaborations with leading University health researchers. He is currently working with Professor Hubert Hondermark on a project to detect metastatic potential in breast cancer cells and with Conjoint
Professor Jim Denham to determine new biomarkers for prostate cancer. He is also working with members of the translational stroke research team on a project investigating indicators of stroke in cerebral spinal fluid.

Baker holds a National Health and Medical Research Council (NHMRC) Career Development Fellowship to research sperm motility and is an investigator on several projects supported by the NHMRC. Late last year he received the 2011 Early Career Researcher Award sponsored by the Hunter Medical Research Institute
(HMRI) fundraising group PULSE.

Baker's research in sperm-egg recognition and proteomic analysis of sperm cells is internationally recognised and has been cited more than 950 times. The proteomics laboratory he was instrumental in establishing, with its world-class mass spectrometry equipment, has made a broad contribution
to research at the University of Newcastle, supporting work across the disciplines of life science, science, medicine and engineering as well as research from HMRI and international and national collaborations.

Two minds are better than one

The powerful combination of Laureate Professor John Aitken's vast knowledge of sperm cell biology and Dr Mark Baker's proteomics prowess makes the University of Newcastle's Reproductive Science Group a formidable force in the research field of sperm function and male fertility.

"John and I decided seven years ago that we needed to investigate the proteins in sperm cells to understand the mechanisms of fertility," Baker advises. "We saw the potential in it and went after it, and the University has backed us all the way. We quickly got into the field and we have raised the
bar."

Baker believes he has benefited greatly from working with Aitken, one of the world's most esteemed reproductive scientists. "He has been my primary mentor, but it is a relationship built on mutual respect," Baker emphasises. "There is a willingness to work together but also a willingness to listen
to each other's points of view – and that is critical to successful research: to be able to agree but also disagree."

Aitken describes Baker as"an ideal example of how postdoctoral researchers can blossom into fully-fledged professional scientists when the University supports their careers."Now, Mark is generating research income in his own right that will more than repay the investment the University has made in
developing his research portfolio."

Career Summary

Biography

After obtaining an Australia postgraduate Award, I did my PhD at Monash University, Department of Biochemistry and Cell Biology under the supervision of Dr. Alfons Lawen. Following a successful time, I was invited to the join the group at the University of Newcastle under Prof. John Aitken, which was a continuation of my PhD theme in the identifying plasma membrane redox systems. Within two years, I decided to set-up and establish a proteomics platform at the University of Newcastle and have since been successful in establishing 4 mass spectrometers in the Advanced mass spectrometry facility. In 2005 I was awarded the university of Newcastle Fellowship (rank #1 from 96 applications) which allowed 5 years of full time research. In 2011 I was awarded the NHMRC career development fellowship (level 2) from the NHMRC. I have been successful on 3 NHMRC DP grants, 1 ARC project grant and 3 ARC LIEF grants.

Research ExpertiseProteomics and phospho-proteomics comparisons using label-free mass spectrometry.

Teaching Expertise6 lectures to Biol 3020 on reproductive physiology.

Administrative ExpertiseGrants, animal ethics, Health and Safety.

CollaborationsNuch Tanphaichitr, Senior Scientist, Ottawa Hospital Research Institute, and Professor in Obstetrics and Gynecology, and Biochemistry/Microbiology/Immunology, University of Ottawa Harvey Florman, University of Massachusetts Melissa Junganickel, University of Massachusetts Gibbes Johnson, Acting Deputy Director, Division of Therapeutic Proteins CDER, FDA, Bethesda University John Aitken, University of Newcastle Neil Sprat, University of Newcastle Tony Velkov, University of Newcastle Brett Nixon, University of Newcastle

Qualifications

PhD, Monash University

Keywords

Biol 3000

Fertilization

Phosphorylation

Spermatozoa

Fields of Research

Code

Description

Percentage

060111

Signal Transduction

40

060199

Biochemistry and Cell Biology not elsewhere classified

30

111499

Paediatrics and Reproductive Medicine not elsewhere classified

30

Professional Experience

UON Appointment

Title

Organisation / Department

Research Fellow

University of NewcastleSchool of Environmental and Life SciencesAustralia

Academic appointment

Dates

Title

Organisation / Department

1/02/2012 - 1/12/2015

Fellow - NHMRC

University of NewcastleSchool of Environmental and Life SciencesAustralia

1/01/2010 -

NHMRC Career Development Fellow

University of NewcastleSchool of Environmental and Life SciencesAustralia

1/06/2007 - 1/02/2012

Fellow - UON

University of NewcastleSchool of Environmental and Life SciencesAustralia

1/01/2005 - 1/01/2011

University of Newcastle Research Fellow

University of NewcastleSchool of Environmental and Life SciencesAustralia

1/01/2002 - 1/01/2005

Post doc

University of NewcastleSchool of Elect Engineering and Computer ScienceAustralia

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell b... [more]

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Porin isoform 1 or VDAC (voltage-dependent anion-selective channel) 1 is the predominant protein in the outer mitochondrial membrane. We demonstrated previously that a plasma memb... [more]

Porin isoform 1 or VDAC (voltage-dependent anion-selective channel) 1 is the predominant protein in the outer mitochondrial membrane. We demonstrated previously that a plasma membrane NADH-ferricyanide reductase activity becomes up-regulated upon mitochondrial perturbation, and therefore suggested that it functions as a cellular redox sensor. VDAC1 is known to be expressed in the plasma membrane; however, its function there remained a mystery. Here we show that VDAC1, when expressed in the plasma membrane, functions as a NADH-ferricyanide reductase. VDAC1 preparations purified from both plasma membrane and mitochondria fractions exhibit NADH-ferricyanide reductase activity, which can be immunoprecipitated with poly- and monoclonal antibodies directed against VDAC(1). Transfecting cells with pl-VDAC1. GFP, which carries an N-terminal signal peptide, directs VDAC1 to the plasma membrane, as shown by confocal microscopy and FACS analysis, and significantly increases the plasma membrane NADH-ferricyanide reductase activity of the transfected cells. This novel enzymatic activity of the well known VDAC1 molecule may provide an explanation for its role in the plasma membrane. Our data suggest that a major function of VDAC1 in the plasma membrane is that of a NADH(-ferricyanide) reductase that may be involved in the maintenance of cellular redox homeostasis.

Research Supervision

Number of supervisions

Total current UON EFTSL

Current Supervision

Determining Factors within Cerebrospinal Fluid that Cause Intracranial Pressure to Rise Post-StrokePhD (Human Physiology), Faculty of Health and Medicine, The University of NewcastleCo-Supervisor

Past Supervision

Year

Level of Study

Research Title / Program / Supervisor Type

2016

PhD

Generation of Gene Disrupted Mice to Further Elucidate the Reproductive Mechanisms of Male Factor FertilityPhD (Biological Sciences), Faculty of Science and Information Technology, The University of NewcastleCo-Supervisor

News

The University of Newcastle (UON) is delighted to announce the following successful researchers in the latest round of National Health and Medical Research Council funding. With the help of this funding, our researchers aim to tackle a range of health-related issues that impact our communities.

Dr Mark Baker has been awarded more than $411,000
in NHMRC Project Grant funding commencing in 2016 for his research
project Understanding idiopathic male infertility: Biomarkers to assist in the diagnosis and assisted reproductive technology outcomes.

Associate Professor Mark Baker

Position

Research FellowSchool of Environmental and Life SciencesFaculty of Science and Information Technology